Aeromonas is an emerging human pathogen, which causes gastroenteritis and septicemia. The organism is being isolated in increasing numbers from food and water, and is becoming resistant to chlorination in water and to multiple antibiotics. The focus of this grant is on a cytotoxic enterotoxin (Act)of Aeromonas, which, in addition to causing gastroenteritis, leads to fatal, non-intestinal infections. Based on the data generated during the current funding period, the following specific aims will be addressed. In Aim 1, we will identify a protein/glycoprotein receptor, which appears to be attached to the plasma membrane by a GPI-anchored protein on the intestinal epithelial cell line (T84), to which Act binds, to initiate a signal transduction cascade. These studies will be accomplished by photoaffinity labeling the receptor with Act, by a genetic approach based on a yeast two-hybrid system, and/or by surface plasmon resonance. In Aim 2, we will delineate the signal transduction cascade triggered by binding of Act to its cell surface receptor on the host cell to better understand the mechanism of action of Act. These studies will involve examining the effect of calcium mobilization and oxidative-stress pathways on TNFalpha and PGE2 production in Act-stimulated cells. In Aim 3, the intracellular trafficking of Act in the host cell will be examined by using specific inhibitors and the mutated Rab proteins using immunofluorescence/confocal microscopy, and we will study stress-associated protein kinase activation by Act from within the host cell. For these studies, Act will be delivered into T84 cells via lipofection or electroporation to prevent receptor-mediated signaling. In Aim 4, we will dissect the role of various mediators generated via Act signaling that lead to different biological effects of Act. We will specifically examine mechanism of Act-induced apoptosis in macrophages via caspase 9 and the role of various biological mediators in fluid secretion, using both in vitro and in vivo models. These studies will provide valuable information in intervening in the severe pathological sequelae associated with Aeromonas infections in the future.